SipW Gene Mutation Construction Of Bacillus Amyloliquefaciens PEBA20 And Its Affect On Bioflim Formation And Antibacterial Activity

In this research,we mainly analyzed the sipW gene of B.amyloliquefaciens PEBA20 which is isolated from poplar.sipW gene is not only regulate the biofilm formation,but also coordinate the bacteriostatic activity,spore formation and various cellular processes of the bacteria.Homologous recombination method is used to build the missing sipW gene mutation.And then verify the influence of B.amyloliquefaciens PEBA20 on biofilm formation,antibacterial activity and spore formation.The main research results are as follows:1.Connected the connected-plasmid sipWF-sipWB and suicide plasmid PMUTin4 by enzyme digestion,T4 ligase connection,and then we obtained the recombinant plasmid sipWF-sipWB-PMUTin4.Recombinant plasmid was transformated into B.amyloliquefaciens PEBA20 competent cell by electroporation.Erythromycin was used to select the PEBA20 sipW mutant strains.Using B.amyloliquefaciens PEBA20 DNA as a template for PCR to select positive clone,and further determined sipW gene deletion mutant strains by sequencing.2.To observe the biofilm formation dynamic characteristics of the B.amyloliquefaciens PEBA20 wild strains and sipW gene deletion mutant strains on solid-gas interface and liquid-gas interface.The results showed that the wild strain and the mutant strains both have the ability of biofilm formation,but compared with wild strains,complex degree of biofilm of sipW gene deletion mutant strains is degraded.we found that sipW gene deletion mutant strains can not form complex fold structure of biofilm on liquid-gas interface.Therefore,sipW gene may be lead to the ability of biofilm formation degraded even lossed.3.Analyzed the antibiotic activity of B.a myloliquefaciens PEBA20 wild strains and sipW mutant strains with three fungi and three bacterial.The results showed that compared with the wild strains,the antimicrobial effect of sipW mutant strains about fungi and bacterial are all decreased,and the significant difference level of some fungi and bacteria is more remarkable.This suggests that the sipW gene mutations may affect the antibacterial activity of biocontrol bacteria about fungi and bacteria.4.Analyzed the morphological characteristics of thallus and spore.The results showed that sipW gene deletion mutation produces spores and mutants with no difference from the wild strain of the bacteria.So sipW gene deletion mutants may not affect spore formation.